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Relationships between dietary, plasma, and muscle amino acids in horses
The Professional Animal Scientist 28 (2012):351–357
©2012 American Registry of Professional Animal Scientists
Relationships between dietary, plasma, and muscle amino acids in horses
P. M. Graham-Thiers,*1 J. A. Wilson,† J. Haught,* and M. Goldberg† *Equine Studies Department, Virginia Intermont College, Bristol 24201; and †Animal Science Department, Berry College, Mount Berry, GA 30149
ABSTRACT Muscle and plasma amino acids were evaluated in horses of various functions. Eight horses were used for exercise (EH) and maintenance (MH) with 5 pregnant mares (PH) that continued into lactation (LH). Five weanlings were used for growth (WH). The EH, MH, and PH groups were used for 8 wk and the LH and WH groups for 4 wk. Blood samples and muscle biopsies were taken at the end of the study and analyzed for amino acids. There were no differences in total muscle amino acids between groups except histidine, which was lower for the EH group. Postfeeding amino acids were greater in the MH, EH, PH, and LH groups for lysine, arginine, and histidine, whereas threonine increased in the MH, EH, and PH groups. The MH group had postfeeding increases in plasma valine, methionine, isoleucine, leucine, and phenylalanine. Correlations within function were found between total and free muscle amino acids as well as plasma for several amino acids. The greatest number of correlations occurred within the weanling group. The WH group had correlations between intake and plasma threonine, lysine, valine, isoleucine, leucine, methionine, and phenylalanine; between intake and total muscle threonine, methionine, 1
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leucine, and phenylalanine; and between plasma and total muscle lysine, phenylalanine, and leucine. These data suggest that the balance between supply and demand of amino acids was in excess for the MH group but close to balance in the WH group. These data suggest potential limiting amino acids for various functions. Key words: amino acid, horse, muscle
INTRODUCTION Specific amino acid requirements for horses are unspecified other than lysine. This limits the ability to improve protein quality for the horse. There is reason to believe differences would exist in amino acid requirements based on the different physiological stresses associated with pregnant, lactating, growing, and even exercising horses. Researchers studying other species such as poultry and swine have developed the concept of amino acid balance by examining the ideal relationships between amino acids through observation of the amino acid balance found in the end products of protein synthesis such as muscle tissue and milk protein (NRC, 2001a,b). Studies in other species have indicated improved production via the use of ideal protein in the dietary supply of amino
acids (Fuller et al., 1989). A better understanding of the amino acid relationships in the horse is needed to optimize dietary supplementation. Previous research has evaluated plasma, muscle, and milk amino acids in horses of various functions, and some correlations have been revealed (Graham-Thiers et al., 2010a,b). The data were in good agreement with previously published data on amino acid concentrations in equine muscle and milk (Bryden, 1991; Wickens et al., 2002). The current study expands on the initial case studies and further evaluates these relationships.
MATERIALS AND METHODS Horses Twenty-one mature horses were used in the study in various functions along with 5 weanlings. Of the mature horses, 8 horses were exercising (EH, geldings, 14.2 ± 2.9 yr, 640.3 ± 18 kg), 8 were in maintenance (MH, geldings, 15.3 ± 1.7 yr, 605.4 ± 17 kg), and 5 were pregnant mares (PH, 12.2 ± 2.6 yr weighing 625.5 ± 14.2 kg). The PH used for the study continued into lactation and became the lactation group (LH). The 5 weanlings (WH) averaged 132 d of age and 186.3 ± 2.1 kg. The EH and MH horses were housed in 3.7 × 3.7
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Table 1. Amino acid composition of commercial grain mixes and hay fed to the maintenance (MH), exercising (EH), pregnant (PH), lactating (LH), and weanling (WH) horses (DM basis) Grain Item CP, % Amino acid, % Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
Hay
MH and EH
PH, LH, and WH
Timothy
Bermudagrass
Alfalfa
16.00 1.01 0.41 0.52 1.04 0.98 0.24 0.71 0.53 0.72
19.80 1.18 0.46 0.72 1.48 0.89 0.30 0.92 0.70 0.86
8.20 0.32 0.11 0.26 0.52 0.28 0.12 0.36 0.28 0.35
12.40 0.47 0.17 0.39 0.78 0.48 0.18 0.48 0.41 0.52
18.90 0.85 0.36 0.77 1.38 0.83 0.27 0.96 0.78 0.98
m box stalls during the day and in a 1.2-ha dry lot at night. The PM, LM, and WH groups were housed in 1.2-ha paddocks as a group and were fed individually. The PH began the study at approximately d 270 of gestation, whereas the LH group continued in the study until approximately 30 d postparturition. The EH group participated in 1 to 2 h of light to moderate work under saddle 5 d each week during the study. All horses received routine preventive health care (deworming, vaccinations) if the schedule for the procedure fell during the study period. The study period lasted for 8 wk for MH, EH, and PH. Although the same horses, the LH group was on the study for 4 wk into lactation. Finally, the WH group was also on the study for 4 wk. The protocol followed the procedures of the Institutional Animal Care and Use Committee in the care and use of the animals throughout the study.
Diets Horses were fed according to current NRC (2007) requirements based on their BW and function. The EH and MH groups were fed a commercial grain mix (Omolene 100, Purina, St. Louis, MO) and a grass hay (timothy/orchardgrass mix). During pregnancy and lactation, mares were fed a commercial grain mix (Mare & Foal, Southern States, Richmond, VA) and
a mostly grass hay (bermudagrass, 20 to 30% alfalfa). Weanlings were fed the same grain mix but were offered hay at a 50:50 ratio of bermudagrass to alfalfa. The nutrient composition of the grain mixes and hays are shown in Table 1. The quantities of each feed offered to the horses were based on recommendations for their specific function to maintain BW in the case of maintenance and exercise, and to support normal fetal development and milk production in the case of pregnant and lactating mares, respectively. Weanlings were fed to support normal ADG for their age. Refusals were noted and weighed daily.
Sampling and Analysis Body weight and BCS were evaluated every 2 wk during the study. Body weight was measured using an electronic scale (model AL660-LA, Cambridge Scaleworks, Honey Brook, PA), whereas BCS was evaluated on a 1 to 9 scale using a standardized system described previously for horses (Henneke et al., 1983). At the end of the study, horses were fasted overnight and a prefeeding blood sample was taken via jugular puncture. Blood samples were drawn again 3 to 4 h after feeding the morning meal for a postfeeding comparison. The timeframe for blood sampling was based on previous evidence that in relation to feeding, urea-N, and
amino acids peak in plasma; 3 to 4 h after feeding (Eggum, 1970, Russell et al., 1986). Plasma was separated and frozen for later analyses of amino acids. At the conclusion of the study, muscle biopsies were taken from the semimembranosus muscle approximately 13 cm below the point of the buttock and 3 to 4 cm deep. A small incision was made through the skin, and approximately 500 mg of muscle was removed. Muscle was immediately frozen in liquid nitrogen for later analyses of free and total amino acids. Muscle samples were homogenized in 0.02 N HCl containing 3.75% sulfosalicylic acid with an internal standard. Muscle protein was removed by centrifugation followed by 0.22-µm filtration, and the remainder was analyzed for free amino acids. Milk samples were taken from mares on d 30 of lactation and frozen for later analysis of free and total amino acids. Amino acid analysis was performed using HPLC (Hitachi L-8800A amino acid analyzer, HTA Corporate, Schaumburg, IL) following the procedure described by Miller-Graber et al. (1990) with concentrations expressed as millimoles per liter for plasma and milk and in millimoles per kilogram of dry muscle for muscle data. Data were summarized as least squares means with SE and were analyzed using the GLM procedure of
Amino acids in horses in various functions
Table 2. Amino acid intake for maintenance (MH), exercising (EH), pregnancy (PH), lactating (LH), and weanling (WH) horses Amino acid
MH, g/d
EH, g/d
PH, g/d
LH, g/d
WH, g/d
SE
42 16 29 58 39 14 40 31 39
56 25 39 76 51 20 53 41 52
99 38 70 139 84 29 88 70 87
114 44 81 161 98 34 102 82 102
26 10 18 36 22 8 23 18 23
2 1 2 3 2 1 2 2 2
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
SAS (SAS Institute Inc., Cary, NC). Comparisons were made between preand postfeeding values for plasma amino acid concentrations within function. The percent change from prefeeding plasma concentrations was compared between functions. Intakes of amino acids expressed on a BW basis (g/kg BW) were used as covariates for between-function comparisons because of differences in amino acid intake. Correlations were also estimated to determine relationships between treatments and measured variables using the CORR procedure in SAS (SAS Institute Inc.). The plasma data used for correlations were taken in the postfeeding state for all horses. Statistical significance was set at a level P < 0.05.
RESULTS AND DISCUSSION All horses with the exception of the WH group maintained BW and BCS. The WH had normal ADG (1.4 ± 0.08 kg/d) and expected BW changes over the course of the study. The PH group consumed 5.0 ± 0.6 kg of the grain mix and 7.2 ± 0.1 kg hay. The LH group consumed 5.6 ± 0.2 kg of the grain mix and 8.1 ± 0.1 kg hay. The WH group consumed 1.3 ± 0.2 kg of the grain mix and 1.7 ± 0.1 kg of the grass:alfalfa hay mix. The EH group consumed 3.7 ± 0.3 kg of the grain mix and 9.1 ± 0.3 kg hay, whereas the MH group consumed 2.1 ± 0.2 kg of the grain mix and 7.8 ± 0.5 kg hay. The intakes of amino acids are shown in Table 2.
Table 3. Muscle amino acid concentrations (total) for maintenance (MH), exercising (EH), pregnancy (PH), lactating (LH), and weanling (WH) horses Amino acid
MH, mmol/kg
EH, mmol/kg
PH, mmol/kg
LH, mmol/kg
WH, mmol/kg
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
34.4 ± 1 28.5 ± 1b 28.3 ± 1 61.7 ± 2 53.4 ± 2 15.3 ± 1 24.9 ± 1 35.1 ± 1 40.7 ± 1
34.8 ± 1 20.0 ± 1a 30.5 ± 2 61.6 ± 2 55.1 ± 2 16.6 ± 1 24.4 ± 1 35.8 ± 1 40.9 ± 1
36.0 ± 2 30.1 ± 1b 26.3 ± 4 59.9 ± 3 55.3 ± 2 14.6 ± 1 23.9 ± 1 35.5 ± 2 38.2 ± 2
38.7 ± 2 28.1 ± 1b 29.2 ± 2 61.1 ± 3 56.3 ± 2 17.0 ± 1 24.5 ± 1 36.3 ± 2 39.9 ± 2
37.4 ± 2 24.3 ± 1b 26.6 ± 4 59.3 ± 3 55.8 ± 2 17.1 ± 1 23.2 ± 1 35.8 ± 2 38.2 ± 2
a,b
Superscripts in the same row are different, P < 0.03.
353 Total muscle concentrations of histidine were lower for the EH group compared with all other groups (P < 0.03). There were no other differences in total muscle amino acid concentrations between groups. The total muscle amino acid data are shown in Table 3. Expressing total muscle amino acid concentrations in relation to lysine (lysine = 100) produced a lower ratio to lysine for histidine in the EH group compared with all other groups. There were no other differences in muscle amino acid ratios. The total muscle amino acid ratios are shown in Table 4. These muscle amino acid ratios are in good agreement with those previously published by Bryden (1991). The difference in total muscle histidine concentration and ratio between the EH group and all other groups may be explained by training increases in muscle carnosine with histidine being a component of carnosine structure. Muscle carnosine was higher for the EH groups compared with all other groups. (P < 0.02; EH = 143.4 ± 23 mmol/ kg). While this is speculation, muscle buffering capacity is increased with training and the major contributor to skeletal muscle buffering is carnosine (Sewell et al., 1991). Also, a correlation has been observed between muscle carnosine content and muscle hypertrophy, which would be expected with training (Sewell et al., 1992). Free muscle amino acids were higher for the EH group compared with all other groups for leucine (P < 0.05). The MH and EH groups had higher free amino acid concentrations of methionine (P < 0.05) compared with all other groups. Free amino acid concentrations of histidine were higher for the EH group compared with the MH and WH group but not the PH or LH group (P < 0.05). The LH group had higher free amino acid concentrations in muscle of lysine compared with PH or WH groups but not EH or MH groups (P < 0.05). There were no other differences between groups for free amino acid concentrations in muscle. The data for free muscle amino acid are shown in Table 5.
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Table 4. Muscle amino acid ratios in relation to lysine1 for maintenance (MH), exercising (EH), pregnant (PH), lactating (LH), and weanling (WH) horses Amino acid
MH
EH
PH
LH
WH
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
64 53b 52 114 100 29 46 65 75
63 35a 54 111 100 30 44 64 73
65 55b 45 109 100 26 43 64 69
69 51b 50 108 100 30 43 64 70
66 43b 57 107 100 30 42 64 69
Superscripts in the same row are different, P < 0.01. Lysine set at 100; all others compared with lysine.
a,b 1
Pre- vs. postfeeding plasma amino acid concentrations were significantly elevated in the MH group for threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, arginine, and histidine (P < 0.05). Pre- vs. postfeeding plasma amino acid concentrations were elevated in the EH and PH group for threonine, lysine, arginine, and histidine (P < 0.05). Pre- vs. postfeeding plasma amino acid concentrations were elevated in the LH group for lysine, arginine, and histidine (P < 0.05). The WH group did not have any significant postfeeding plasma amino acid changes. Comparing the percent change from prefeeding plasma amino acid concentrations between groups,
the MH group had significantly greater percent changes for arginine, isoleucine, lysine, methionine, and threonine compared with all other groups (P < 0.05). The MH group also had greater percent changes in histidine compared with the WH group but not the other groups (P < 0.05). The percent changes in plasma leucine and phenylalanine concentrations were greater for the MH group compared with the EH, PH, and WH groups but not the LH group (P < 0.05). The percent changes from prefeeding for plasma amino acid concentrations are shown in Table 6. Plasma amino acid concentrations are a reflection of influx (from gut and tissue) and efflux (removal by
Figure 1. Correlations between intake of phenylalanine on a BW basis per day and plasma concentrations of phenylalanine (r = −0.92, P = 0.026) as well as with total muscle concentrations of phenylalanine (r = 0.96, P = 0.04) for the lactating horses.
tissues), thus reflecting a net “balance” of the respective amino acid concentration. With this in mind, one may speculate on the amino acid status of the animal based on amino acid concentrations at key times such as times when peak concentrations are expected. Research in other species has demonstrated that amino acid concentrations, which increase the least in response to feeding after a fast, indicates the limiting amino acid for that animal (Potter et al., 1972). Although the use of isotopes and tracers has made this outdated for many species, use of tracer techniques is rare in horses. When evaluating these “balances,” it appears that the MH group consumed amino acids well in excess of demands and the WH had high demands compared with the
Table 5. Muscle free amino acid concentrations for maintenance (MH), exercising (EH), pregnant (PH), lactating (LH), and weanling (WH) horses Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
EH, mmol/kg
PH, mmol/kg
LH, mmol/kg
WH, mmol/kg
ND1 0.06 ± 0.07b 0.23 ± 0.04 0.07 ± 0.07b 0.39 ± 0.16a 0.42 ± 0.16a ND 0.24 ± 0.2 0.14 ± 0.012
0.23 ± 0.06 0.23 ± 0.05a 0.23 ± 0.04 0.30 ± 0.07a 0.64 ± 0.15a 0.36 ± 0.14a 0.13 ± 0.07 0.40 ± 0.20 0.15 ± 0.12
0.08 ± 0.01 0.15 ± 0.04ab 0.03 ± 0.03 0.09 ± 0.07b 0.06 ± 0.06b 0.08 ± 0.05b 0.16 ± 0.04 0.02 ± 0.05 0.14 ± 0.04
ND 0.13 ± 0.06ab 0.02 ± 0.01 0.11 ± 0.09b 0.36 ± 0.21a 0.18 ± 0.08b 0.22 ± 0.06 0.01 ± 0.01 0.11 ± 0.13
0.01 ± 0.01 0.08 ± 0.04b 0.01 ± 0.02 0.08 ± 0.05b 0.11 ± 0.12b 0.03 ± 0.03b 0.13 ± 0.03 0.08 ± 0.05 0.13 ± 0.19
Superscripts in the same row are different, P < 0.05. ND = not detected.
a,b 1
MH, mmol/kg
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Amino acids in horses in various functions
Table 6. Percent change from fasting plasma amino acid concentrations for maintenance (MH), exercising (EH), pregnant (PH), lactating (LH), and weanling (WH) horses Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine a,b
MH, mmol/L
EH, mmol/L
PH, mmol/L
LH, mmol/L
WH, mmol/L
103.5 ± 13b 38.5 ± 9b 65.0 ± 17b 48.1 ± 12b 104.3 ± 12c 88.4 ± 20b 31.9 ± 7b 74.3 ± 10b 42.1 ± 15
40.1 ± 14a 31.5 ± 10ab −3.8 ± 18a −1.4 ± 14a 48.3 ± 13b 12.6 ± 21a 7.0 ± 7a 36.4 ± 11a 15.4 ± 16
40.1 ± 16a 21.4 ± 11ab 3.3 ± 21a 3.0 ± 16a 44.4 ± 15b 4.0 ± 25a 8.3 ± 9a 30.1 ± 13a 27.9 ± 19
43.4 ± 16a 30.5 ± 11ab 20.1 ± 21ab 46.1 ± 16b 40.3 ± 15b 7.1 ± 25a 18.0 ± 9ab 31.0 ± 13a 59.3 ± 19
11.9 ± 16a 8.3 ± 11ab 6.5 ± 21a 3.7 ± 16a 1.4 ± 15b 0.1 ± 25a 9.1 ± 9a 7.2 ± 13a 10.2 ± 19
Superscripts in the same row are different, P < 0.05.
supply based on a lack of significant increases in the postfeeding amino acid concentrations. Reviewing the percent changes within functions and observing the smallest change in pre- vs. postfeeding concentrations, the limiting amino acids for the WH group would be methionine and lysine and for the LH it would be methionine. These are not surprising because lysine has been shown to be the first limiting amino acid for growth in the horse (Ott et al., 1979) and methionine has been shown to be limiting for milk production in the dairy cow (NRC, 2001a,b). Following the same premise, the MH group would be limiting in phenylalanine. Although the percent change was the smallest relative to the other amino acids for the MH group, it was not that small (~32%). Interestingly, the limiting amino acid for both the EH and PH
group would be isoleucine and leucine based on the fact that plasma concentrations decreased postfeeding in the EH group. Correlations were found for PH group between intake and plasma phenylalanine (r = −0.95, P = 0.012). Correlations were also found between plasma and free muscle concentrations of leucine and histidine (r = 0.95, P = 0.014 and r = 0.83, P = 0.05, respectively) for the PH group. The negative correlations and lack of many other correlations may suggest demand for amino acids in other tissues to be more important in this function such as fetal tissue. The LH group had correlations between intake and total muscle concentrations for valine (r = 0.98, P = 0.016) and phenylalanine (r = 0.96, P = 0.04; Figure 1). There was a negative correlation between intake
and plasma phenylalanine for the LH group (r = −0.92, P = 0.026; Figure 1). Muscle and plasma concentrations of phenylalanine were also negatively correlated for the LH group (r = −0.95, P = 0.05; Figure 2). Plasma and free muscle concentrations of valine were correlated for the LH group (r = 0.97, P = 0.029). Total muscle concentrations of histidine and total milk concentrations of histidine were negatively correlated for the LH group (r = −0.97, P = 0.023), whereas total muscle concentrations of leucine were correlated to free milk concentrations of leucine for the LH group (r = 0.99, P = 0.0063). The numerous negative correlations between plasma and muscle amino acid concentrations may suggest a catabolic state for the lactating mare with the mammary gland taking precedence over muscle tissue. The positive correlations between
Figure 2. Correlation between plasma concentrations of phenylalanine and total muscle concentrations of phenylalanine for the lactating horses (r = −0.95, P = 0.05).
Figure 3. Correlations between intake of threonine on a BW basis per day and plasma concentrations of threonine (r = 0.90, P = 0.036) as well as total muscle concentrations of threonine (r = 0.90, P = 0.035) for the weanling horses.
Figure 4. Correlations between intake of lysine on a BW basis per day and plasma concentrations of lysine (r = 0.92, P = 0.027) as well as total muscle concentrations of lysine (r = 0.87, P = 0.05) for the weanling horses.
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Figure 5. Correlations between intake of leucine on a BW basis per day and plasma leucine concentrations (r = 0.93, P = 0.021) as well as total muscle concentrations (r = 0.88, P = 0.046) for the weanling horses.
plasma and free muscle amino acids along with the positive correlation between muscle and milk amino acid concentrations seems to support the priority for amino acid supply to milk. The WH group had several correlations for intake and plasma amino acids concentrations including threonine (r = 0.90, P = 0.036; Figure 3), lysine (r = 0.92, P = 0.027; Figure 4), valine (r = 0.89, P = 0.042), isoleucine (r = 0.96, P = 0.0083), leucine (P = 0.021; Figure 5), arginine (r = 0.93, P = 0.021), methionine (r = 0.89, P = 0.042; Figure 6), and phenylalanine (r = 0.90, P = 0.035; Figure 7). Intake and total muscle amino acids concentrations were correlated in the WH groups for phenylalanine (r = 0.88, P = 0.049; Figure 7), lysine (r = 0.87, P = 0.05; Figure 4), threonine (r = 0.90, P = 0.035; Figure 3), methionine (r = 0.87, P = 0.05; Figure 6), and leucine (r = 0.88, P
Figure 6. Correlations between intake of methionine on a BW basis per day and plasma concentrations of methionine (r = 0.89, P = 0.042) as well as total muscle concentrations of methionine (r = 0.87, P = 0.05) for the weanling horses.
Graham-Thiers et al.
Figure 7. Correlations between intake of phenylalanine on a BW basis per day and plasma concentrations of phenylalanine (r = 0.90, P = 0.035) as well as total muscle concentrations of phenylalanine (r = 0.88, P = 0.049) for the weanling horses.
Figure 9. Correlation of plasma concentrations of phenylalanine and total muscle concentrations of phenylalanine for the weanling horses (r = 0.92, P = 0.028).
= 0.046; Figure 5). Plasma and total muscle amino acids concentrations were correlated for lysine (r = 0.99, P = 0.0013; Figure 8), phenylalanine (r = 0.92, P = 0.028; Figure 9), and leucine in the WH group (r = 0.98, P = 0.0034; Figure 10). The large number of correlations between plasma and muscle amino acid concentrations observed in the WH group support the conclusion that the supply of amino acids closely reflected the subsequent demand for those amino acids by the growing horse. This could also be indicative of an anabolic state in which growth would be considered an anabolic function. The EH group had a negative correlation between total muscle amino acid concentrations and free amino acid muscle concentrations of histidine (r = −0.88, P = 0.0098) and between plasma and total muscle concentrations of phenylalanine (r = −0.82, P = 0.024; Figure 11). As stated previ-
ously, negative correlations between amino acid concentrations in plasma and muscle may suggest that other tissues may be taking priority over muscle tissue in the current function. This would not seem to be warranted in the exercising horse and may suggest catabolic state for the EH when the samples were taken. This is a limitation to taking single samples and admittedly may not represent the ongoing state of the EH. The MH group had negative correlations between intake and free muscle amino acid concentrations of lysine (r = −0.74, P = 0.037) as well as between plasma and total muscle amino acid concentrations of arginine (r = −0.89, P = 0.003; Figure 12). No other correlations were observed. The lack of correlations in the MH group could therefore also support the conclusion that the amino acid supply did not reflect the demand by the MH group, which is also supported by the large increases in plasma amino acid
Figure 8. Correlation of plasma concentrations of lysine and total muscle concentrations of lysine for the weanling horses (r = 0.99, P = 0.0013).
Figure 10. Correlation of plasma concentrations of leucine and total muscle concentrations of leucine for the weanling horses (r = 0.98, P = 0.0034).
Amino acids in horses in various functions
Fuller, M. F., R. McWilliam, T. C. Wang, and L. R. Giles. 1989. The optimum dietary amino acid pattern for growing pigs. 2. Requirements for maintenance and tissue accretion. Br. J. Nutr. 62:255–267.
Table 7. Total and free milk amino acids for LH mares at d 30 of lactation Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine 1 2
Free amino acids, mmol/L
Total amino acids, mmol/L
Ratio1
0.07 ± 0.015 0.30 ± 0.065 0.09 ± 0.010 0.19 ± 0.040 0.12 ± 0.025 ND2 0.10 ± 0.025 0.11 ± 0.017 0.42 ± 0.050
25.1 ± 1.3 12.9 ± 0.7 26.8 ± 1.9 54.5 ± 2.8 40.2 ± 2.6 10.9 ± 0.6 20.1 ± 1.1 32.3 ± 1.8 37.6 ± 1.6
62 32 67 136 100 27 50 80 94
Lysine set at 100; all others compared with lysine. ND = not detected.
IMPLICATIONS
Figure 12. Correlation between plasma concentrations of arginine and total muscle concentrations of arginine for the maintenance horses (r = −0.89, P = 0.003).
concentrations in this group postfeeding, thus suggesting excesses. The free and total milk amino acids are reported in Table 7. The total milk amino acid ratios were calculated in relation to lysine. These ratios
Graham-Thiers, P. M., J. A. Wilson, J. Haught, and M. Goldberg. 2010a. Case Study: Relationships between dietary, plasma, and muscle amino acids in maintenance horses, exercising horses, and growing weanling horses. Prof. Anim. Sci. 26:313–319. Graham-Thiers, P. M., J. A. Wilson, J. Haught, and M. Goldberg. 2010b. Case Study: Relationships between dietary, plasma, and muscle amino acids in maintenance, pregnant, and lactating mares. Prof. Anim. Sci. 26:320–327. Henneke, D. R., G. D. Potter, J. L. Kreider, and B. F. Yeates. 1983. Relationship between condition score, physical measurements and body fat percentage in mares. Equine Vet. J. 15:371–372.
are in good agreement with other published reports of milk amino acid ratios (Bryden, 1991; Wickens et al., 2002).
Figure 11. Correlation of plasma concentrations of phenylalanine and total muscle concentrations of phenylalanine for the exercising horses (r = −0.82, P = 0.024).
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It appears from these data that for the most part, the body does not alter the muscle amino acid pattern regardless of the demands on the body. The supply of amino acids from plasma and muscle free amino acids does vary depending on function (as well as intake). The authors want to reiterate, however, that the data of the current study provide information only for the time point in which the plasma was sampled. The data may appear different if taken at another time point. These data provide additional information regarding amino acids in the equine, particularly the total muscle amino acid profile as well as the milk amino acid profile for which there is limited data. These data could assist in the development of an ideal protein concept for the horse.
LITERATURE CITED Bryden, W. L. 1991. Amino acid requirements of horses estimated from tissue composition. Page 53 in Proc. Nutr. Soc. Aust. HEC Press, Danvers, MA. Eggum, B. O. 1970. Blood urea measurement as a technique for assessing protein quality. Br. J. Nutr. 24:983–988.
Miller-Graber, P. A., L. M. Lawrence, E. Kurcz, R. Kane, K. Bump, M. Fisher, and J. Smith. 1990. The free amino acid profile in the middle gluteal before and after fatiguing exercise in the horse. Equine Vet. J. 22:209–210. NRC. 2001a. Nutrient Requirements of Dairy Cattle. 7th ed. Natl. Acad. Press, Washington, DC. NRC. 2001b. Nutrient Requirements of Swine. 10th ed. Natl. Acad. Press, Washington, DC. NRC. 2007. Nutrient Requirements of Horses. 6th ed. Natl. Acad. Press, Washington, DC. Ott, E. A., R. L. Asquith, J. P. Feaster, and F. G. Martin. 1979. Influence of protein level and quality on growth and development of yearling foals. J. Anim. Sci. 49:620–626. Potter, E. L., D. B. Purser, and W. G. Bergen. 1972. A plasma reference index for predicting limiting amino acids of sheep and rats. J. Anim. Sci. 34:660–671. Russell, M. A., A. V. Rodiek, and L. M. Lawrence. 1986. Effect of meal schedules and fasting on selected plasma free amino acids in horses. J. Anim. Sci. 63:1428–1431. Sewell, D. A., R. C. Harris, and M. Dunnett. 1991. Carnosine accounts for most of the variation in the physic-chemical buffering in equine muscle. Equine Exercise Physiol. 3:276–280. Sewell, D. A., R. C. Harris, D. J. Marlin, and M. Dunnett. 1992. Estimation of the carnosine content of different fibre types in the middle gluteal muscle of the Thoroughbred horse. J. Physiol. 455:447–453. Wickens, C. L., P. K. Ku, and N. L. Trottier. 2002. An ideal protein for the lactating mare. J. Anim. Sci. 80(Suppl. 1):155.
©2012 American Registry of Professional Animal Scientists
Relationships between dietary, plasma, and muscle amino acids in horses
P. M. Graham-Thiers,*1 J. A. Wilson,† J. Haught,* and M. Goldberg† *Equine Studies Department, Virginia Intermont College, Bristol 24201; and †Animal Science Department, Berry College, Mount Berry, GA 30149
ABSTRACT Muscle and plasma amino acids were evaluated in horses of various functions. Eight horses were used for exercise (EH) and maintenance (MH) with 5 pregnant mares (PH) that continued into lactation (LH). Five weanlings were used for growth (WH). The EH, MH, and PH groups were used for 8 wk and the LH and WH groups for 4 wk. Blood samples and muscle biopsies were taken at the end of the study and analyzed for amino acids. There were no differences in total muscle amino acids between groups except histidine, which was lower for the EH group. Postfeeding amino acids were greater in the MH, EH, PH, and LH groups for lysine, arginine, and histidine, whereas threonine increased in the MH, EH, and PH groups. The MH group had postfeeding increases in plasma valine, methionine, isoleucine, leucine, and phenylalanine. Correlations within function were found between total and free muscle amino acids as well as plasma for several amino acids. The greatest number of correlations occurred within the weanling group. The WH group had correlations between intake and plasma threonine, lysine, valine, isoleucine, leucine, methionine, and phenylalanine; between intake and total muscle threonine, methionine, 1
Corresponding author: [email protected]
leucine, and phenylalanine; and between plasma and total muscle lysine, phenylalanine, and leucine. These data suggest that the balance between supply and demand of amino acids was in excess for the MH group but close to balance in the WH group. These data suggest potential limiting amino acids for various functions. Key words: amino acid, horse, muscle
INTRODUCTION Specific amino acid requirements for horses are unspecified other than lysine. This limits the ability to improve protein quality for the horse. There is reason to believe differences would exist in amino acid requirements based on the different physiological stresses associated with pregnant, lactating, growing, and even exercising horses. Researchers studying other species such as poultry and swine have developed the concept of amino acid balance by examining the ideal relationships between amino acids through observation of the amino acid balance found in the end products of protein synthesis such as muscle tissue and milk protein (NRC, 2001a,b). Studies in other species have indicated improved production via the use of ideal protein in the dietary supply of amino
acids (Fuller et al., 1989). A better understanding of the amino acid relationships in the horse is needed to optimize dietary supplementation. Previous research has evaluated plasma, muscle, and milk amino acids in horses of various functions, and some correlations have been revealed (Graham-Thiers et al., 2010a,b). The data were in good agreement with previously published data on amino acid concentrations in equine muscle and milk (Bryden, 1991; Wickens et al., 2002). The current study expands on the initial case studies and further evaluates these relationships.
MATERIALS AND METHODS Horses Twenty-one mature horses were used in the study in various functions along with 5 weanlings. Of the mature horses, 8 horses were exercising (EH, geldings, 14.2 ± 2.9 yr, 640.3 ± 18 kg), 8 were in maintenance (MH, geldings, 15.3 ± 1.7 yr, 605.4 ± 17 kg), and 5 were pregnant mares (PH, 12.2 ± 2.6 yr weighing 625.5 ± 14.2 kg). The PH used for the study continued into lactation and became the lactation group (LH). The 5 weanlings (WH) averaged 132 d of age and 186.3 ± 2.1 kg. The EH and MH horses were housed in 3.7 × 3.7
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Table 1. Amino acid composition of commercial grain mixes and hay fed to the maintenance (MH), exercising (EH), pregnant (PH), lactating (LH), and weanling (WH) horses (DM basis) Grain Item CP, % Amino acid, % Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
Hay
MH and EH
PH, LH, and WH
Timothy
Bermudagrass
Alfalfa
16.00 1.01 0.41 0.52 1.04 0.98 0.24 0.71 0.53 0.72
19.80 1.18 0.46 0.72 1.48 0.89 0.30 0.92 0.70 0.86
8.20 0.32 0.11 0.26 0.52 0.28 0.12 0.36 0.28 0.35
12.40 0.47 0.17 0.39 0.78 0.48 0.18 0.48 0.41 0.52
18.90 0.85 0.36 0.77 1.38 0.83 0.27 0.96 0.78 0.98
m box stalls during the day and in a 1.2-ha dry lot at night. The PM, LM, and WH groups were housed in 1.2-ha paddocks as a group and were fed individually. The PH began the study at approximately d 270 of gestation, whereas the LH group continued in the study until approximately 30 d postparturition. The EH group participated in 1 to 2 h of light to moderate work under saddle 5 d each week during the study. All horses received routine preventive health care (deworming, vaccinations) if the schedule for the procedure fell during the study period. The study period lasted for 8 wk for MH, EH, and PH. Although the same horses, the LH group was on the study for 4 wk into lactation. Finally, the WH group was also on the study for 4 wk. The protocol followed the procedures of the Institutional Animal Care and Use Committee in the care and use of the animals throughout the study.
Diets Horses were fed according to current NRC (2007) requirements based on their BW and function. The EH and MH groups were fed a commercial grain mix (Omolene 100, Purina, St. Louis, MO) and a grass hay (timothy/orchardgrass mix). During pregnancy and lactation, mares were fed a commercial grain mix (Mare & Foal, Southern States, Richmond, VA) and
a mostly grass hay (bermudagrass, 20 to 30% alfalfa). Weanlings were fed the same grain mix but were offered hay at a 50:50 ratio of bermudagrass to alfalfa. The nutrient composition of the grain mixes and hays are shown in Table 1. The quantities of each feed offered to the horses were based on recommendations for their specific function to maintain BW in the case of maintenance and exercise, and to support normal fetal development and milk production in the case of pregnant and lactating mares, respectively. Weanlings were fed to support normal ADG for their age. Refusals were noted and weighed daily.
Sampling and Analysis Body weight and BCS were evaluated every 2 wk during the study. Body weight was measured using an electronic scale (model AL660-LA, Cambridge Scaleworks, Honey Brook, PA), whereas BCS was evaluated on a 1 to 9 scale using a standardized system described previously for horses (Henneke et al., 1983). At the end of the study, horses were fasted overnight and a prefeeding blood sample was taken via jugular puncture. Blood samples were drawn again 3 to 4 h after feeding the morning meal for a postfeeding comparison. The timeframe for blood sampling was based on previous evidence that in relation to feeding, urea-N, and
amino acids peak in plasma; 3 to 4 h after feeding (Eggum, 1970, Russell et al., 1986). Plasma was separated and frozen for later analyses of amino acids. At the conclusion of the study, muscle biopsies were taken from the semimembranosus muscle approximately 13 cm below the point of the buttock and 3 to 4 cm deep. A small incision was made through the skin, and approximately 500 mg of muscle was removed. Muscle was immediately frozen in liquid nitrogen for later analyses of free and total amino acids. Muscle samples were homogenized in 0.02 N HCl containing 3.75% sulfosalicylic acid with an internal standard. Muscle protein was removed by centrifugation followed by 0.22-µm filtration, and the remainder was analyzed for free amino acids. Milk samples were taken from mares on d 30 of lactation and frozen for later analysis of free and total amino acids. Amino acid analysis was performed using HPLC (Hitachi L-8800A amino acid analyzer, HTA Corporate, Schaumburg, IL) following the procedure described by Miller-Graber et al. (1990) with concentrations expressed as millimoles per liter for plasma and milk and in millimoles per kilogram of dry muscle for muscle data. Data were summarized as least squares means with SE and were analyzed using the GLM procedure of
Amino acids in horses in various functions
Table 2. Amino acid intake for maintenance (MH), exercising (EH), pregnancy (PH), lactating (LH), and weanling (WH) horses Amino acid
MH, g/d
EH, g/d
PH, g/d
LH, g/d
WH, g/d
SE
42 16 29 58 39 14 40 31 39
56 25 39 76 51 20 53 41 52
99 38 70 139 84 29 88 70 87
114 44 81 161 98 34 102 82 102
26 10 18 36 22 8 23 18 23
2 1 2 3 2 1 2 2 2
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
SAS (SAS Institute Inc., Cary, NC). Comparisons were made between preand postfeeding values for plasma amino acid concentrations within function. The percent change from prefeeding plasma concentrations was compared between functions. Intakes of amino acids expressed on a BW basis (g/kg BW) were used as covariates for between-function comparisons because of differences in amino acid intake. Correlations were also estimated to determine relationships between treatments and measured variables using the CORR procedure in SAS (SAS Institute Inc.). The plasma data used for correlations were taken in the postfeeding state for all horses. Statistical significance was set at a level P < 0.05.
RESULTS AND DISCUSSION All horses with the exception of the WH group maintained BW and BCS. The WH had normal ADG (1.4 ± 0.08 kg/d) and expected BW changes over the course of the study. The PH group consumed 5.0 ± 0.6 kg of the grain mix and 7.2 ± 0.1 kg hay. The LH group consumed 5.6 ± 0.2 kg of the grain mix and 8.1 ± 0.1 kg hay. The WH group consumed 1.3 ± 0.2 kg of the grain mix and 1.7 ± 0.1 kg of the grass:alfalfa hay mix. The EH group consumed 3.7 ± 0.3 kg of the grain mix and 9.1 ± 0.3 kg hay, whereas the MH group consumed 2.1 ± 0.2 kg of the grain mix and 7.8 ± 0.5 kg hay. The intakes of amino acids are shown in Table 2.
Table 3. Muscle amino acid concentrations (total) for maintenance (MH), exercising (EH), pregnancy (PH), lactating (LH), and weanling (WH) horses Amino acid
MH, mmol/kg
EH, mmol/kg
PH, mmol/kg
LH, mmol/kg
WH, mmol/kg
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
34.4 ± 1 28.5 ± 1b 28.3 ± 1 61.7 ± 2 53.4 ± 2 15.3 ± 1 24.9 ± 1 35.1 ± 1 40.7 ± 1
34.8 ± 1 20.0 ± 1a 30.5 ± 2 61.6 ± 2 55.1 ± 2 16.6 ± 1 24.4 ± 1 35.8 ± 1 40.9 ± 1
36.0 ± 2 30.1 ± 1b 26.3 ± 4 59.9 ± 3 55.3 ± 2 14.6 ± 1 23.9 ± 1 35.5 ± 2 38.2 ± 2
38.7 ± 2 28.1 ± 1b 29.2 ± 2 61.1 ± 3 56.3 ± 2 17.0 ± 1 24.5 ± 1 36.3 ± 2 39.9 ± 2
37.4 ± 2 24.3 ± 1b 26.6 ± 4 59.3 ± 3 55.8 ± 2 17.1 ± 1 23.2 ± 1 35.8 ± 2 38.2 ± 2
a,b
Superscripts in the same row are different, P < 0.03.
353 Total muscle concentrations of histidine were lower for the EH group compared with all other groups (P < 0.03). There were no other differences in total muscle amino acid concentrations between groups. The total muscle amino acid data are shown in Table 3. Expressing total muscle amino acid concentrations in relation to lysine (lysine = 100) produced a lower ratio to lysine for histidine in the EH group compared with all other groups. There were no other differences in muscle amino acid ratios. The total muscle amino acid ratios are shown in Table 4. These muscle amino acid ratios are in good agreement with those previously published by Bryden (1991). The difference in total muscle histidine concentration and ratio between the EH group and all other groups may be explained by training increases in muscle carnosine with histidine being a component of carnosine structure. Muscle carnosine was higher for the EH groups compared with all other groups. (P < 0.02; EH = 143.4 ± 23 mmol/ kg). While this is speculation, muscle buffering capacity is increased with training and the major contributor to skeletal muscle buffering is carnosine (Sewell et al., 1991). Also, a correlation has been observed between muscle carnosine content and muscle hypertrophy, which would be expected with training (Sewell et al., 1992). Free muscle amino acids were higher for the EH group compared with all other groups for leucine (P < 0.05). The MH and EH groups had higher free amino acid concentrations of methionine (P < 0.05) compared with all other groups. Free amino acid concentrations of histidine were higher for the EH group compared with the MH and WH group but not the PH or LH group (P < 0.05). The LH group had higher free amino acid concentrations in muscle of lysine compared with PH or WH groups but not EH or MH groups (P < 0.05). There were no other differences between groups for free amino acid concentrations in muscle. The data for free muscle amino acid are shown in Table 5.
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Table 4. Muscle amino acid ratios in relation to lysine1 for maintenance (MH), exercising (EH), pregnant (PH), lactating (LH), and weanling (WH) horses Amino acid
MH
EH
PH
LH
WH
Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
64 53b 52 114 100 29 46 65 75
63 35a 54 111 100 30 44 64 73
65 55b 45 109 100 26 43 64 69
69 51b 50 108 100 30 43 64 70
66 43b 57 107 100 30 42 64 69
Superscripts in the same row are different, P < 0.01. Lysine set at 100; all others compared with lysine.
a,b 1
Pre- vs. postfeeding plasma amino acid concentrations were significantly elevated in the MH group for threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, arginine, and histidine (P < 0.05). Pre- vs. postfeeding plasma amino acid concentrations were elevated in the EH and PH group for threonine, lysine, arginine, and histidine (P < 0.05). Pre- vs. postfeeding plasma amino acid concentrations were elevated in the LH group for lysine, arginine, and histidine (P < 0.05). The WH group did not have any significant postfeeding plasma amino acid changes. Comparing the percent change from prefeeding plasma amino acid concentrations between groups,
the MH group had significantly greater percent changes for arginine, isoleucine, lysine, methionine, and threonine compared with all other groups (P < 0.05). The MH group also had greater percent changes in histidine compared with the WH group but not the other groups (P < 0.05). The percent changes in plasma leucine and phenylalanine concentrations were greater for the MH group compared with the EH, PH, and WH groups but not the LH group (P < 0.05). The percent changes from prefeeding for plasma amino acid concentrations are shown in Table 6. Plasma amino acid concentrations are a reflection of influx (from gut and tissue) and efflux (removal by
Figure 1. Correlations between intake of phenylalanine on a BW basis per day and plasma concentrations of phenylalanine (r = −0.92, P = 0.026) as well as with total muscle concentrations of phenylalanine (r = 0.96, P = 0.04) for the lactating horses.
tissues), thus reflecting a net “balance” of the respective amino acid concentration. With this in mind, one may speculate on the amino acid status of the animal based on amino acid concentrations at key times such as times when peak concentrations are expected. Research in other species has demonstrated that amino acid concentrations, which increase the least in response to feeding after a fast, indicates the limiting amino acid for that animal (Potter et al., 1972). Although the use of isotopes and tracers has made this outdated for many species, use of tracer techniques is rare in horses. When evaluating these “balances,” it appears that the MH group consumed amino acids well in excess of demands and the WH had high demands compared with the
Table 5. Muscle free amino acid concentrations for maintenance (MH), exercising (EH), pregnant (PH), lactating (LH), and weanling (WH) horses Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
EH, mmol/kg
PH, mmol/kg
LH, mmol/kg
WH, mmol/kg
ND1 0.06 ± 0.07b 0.23 ± 0.04 0.07 ± 0.07b 0.39 ± 0.16a 0.42 ± 0.16a ND 0.24 ± 0.2 0.14 ± 0.012
0.23 ± 0.06 0.23 ± 0.05a 0.23 ± 0.04 0.30 ± 0.07a 0.64 ± 0.15a 0.36 ± 0.14a 0.13 ± 0.07 0.40 ± 0.20 0.15 ± 0.12
0.08 ± 0.01 0.15 ± 0.04ab 0.03 ± 0.03 0.09 ± 0.07b 0.06 ± 0.06b 0.08 ± 0.05b 0.16 ± 0.04 0.02 ± 0.05 0.14 ± 0.04
ND 0.13 ± 0.06ab 0.02 ± 0.01 0.11 ± 0.09b 0.36 ± 0.21a 0.18 ± 0.08b 0.22 ± 0.06 0.01 ± 0.01 0.11 ± 0.13
0.01 ± 0.01 0.08 ± 0.04b 0.01 ± 0.02 0.08 ± 0.05b 0.11 ± 0.12b 0.03 ± 0.03b 0.13 ± 0.03 0.08 ± 0.05 0.13 ± 0.19
Superscripts in the same row are different, P < 0.05. ND = not detected.
a,b 1
MH, mmol/kg
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Amino acids in horses in various functions
Table 6. Percent change from fasting plasma amino acid concentrations for maintenance (MH), exercising (EH), pregnant (PH), lactating (LH), and weanling (WH) horses Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine a,b
MH, mmol/L
EH, mmol/L
PH, mmol/L
LH, mmol/L
WH, mmol/L
103.5 ± 13b 38.5 ± 9b 65.0 ± 17b 48.1 ± 12b 104.3 ± 12c 88.4 ± 20b 31.9 ± 7b 74.3 ± 10b 42.1 ± 15
40.1 ± 14a 31.5 ± 10ab −3.8 ± 18a −1.4 ± 14a 48.3 ± 13b 12.6 ± 21a 7.0 ± 7a 36.4 ± 11a 15.4 ± 16
40.1 ± 16a 21.4 ± 11ab 3.3 ± 21a 3.0 ± 16a 44.4 ± 15b 4.0 ± 25a 8.3 ± 9a 30.1 ± 13a 27.9 ± 19
43.4 ± 16a 30.5 ± 11ab 20.1 ± 21ab 46.1 ± 16b 40.3 ± 15b 7.1 ± 25a 18.0 ± 9ab 31.0 ± 13a 59.3 ± 19
11.9 ± 16a 8.3 ± 11ab 6.5 ± 21a 3.7 ± 16a 1.4 ± 15b 0.1 ± 25a 9.1 ± 9a 7.2 ± 13a 10.2 ± 19
Superscripts in the same row are different, P < 0.05.
supply based on a lack of significant increases in the postfeeding amino acid concentrations. Reviewing the percent changes within functions and observing the smallest change in pre- vs. postfeeding concentrations, the limiting amino acids for the WH group would be methionine and lysine and for the LH it would be methionine. These are not surprising because lysine has been shown to be the first limiting amino acid for growth in the horse (Ott et al., 1979) and methionine has been shown to be limiting for milk production in the dairy cow (NRC, 2001a,b). Following the same premise, the MH group would be limiting in phenylalanine. Although the percent change was the smallest relative to the other amino acids for the MH group, it was not that small (~32%). Interestingly, the limiting amino acid for both the EH and PH
group would be isoleucine and leucine based on the fact that plasma concentrations decreased postfeeding in the EH group. Correlations were found for PH group between intake and plasma phenylalanine (r = −0.95, P = 0.012). Correlations were also found between plasma and free muscle concentrations of leucine and histidine (r = 0.95, P = 0.014 and r = 0.83, P = 0.05, respectively) for the PH group. The negative correlations and lack of many other correlations may suggest demand for amino acids in other tissues to be more important in this function such as fetal tissue. The LH group had correlations between intake and total muscle concentrations for valine (r = 0.98, P = 0.016) and phenylalanine (r = 0.96, P = 0.04; Figure 1). There was a negative correlation between intake
and plasma phenylalanine for the LH group (r = −0.92, P = 0.026; Figure 1). Muscle and plasma concentrations of phenylalanine were also negatively correlated for the LH group (r = −0.95, P = 0.05; Figure 2). Plasma and free muscle concentrations of valine were correlated for the LH group (r = 0.97, P = 0.029). Total muscle concentrations of histidine and total milk concentrations of histidine were negatively correlated for the LH group (r = −0.97, P = 0.023), whereas total muscle concentrations of leucine were correlated to free milk concentrations of leucine for the LH group (r = 0.99, P = 0.0063). The numerous negative correlations between plasma and muscle amino acid concentrations may suggest a catabolic state for the lactating mare with the mammary gland taking precedence over muscle tissue. The positive correlations between
Figure 2. Correlation between plasma concentrations of phenylalanine and total muscle concentrations of phenylalanine for the lactating horses (r = −0.95, P = 0.05).
Figure 3. Correlations between intake of threonine on a BW basis per day and plasma concentrations of threonine (r = 0.90, P = 0.036) as well as total muscle concentrations of threonine (r = 0.90, P = 0.035) for the weanling horses.
Figure 4. Correlations between intake of lysine on a BW basis per day and plasma concentrations of lysine (r = 0.92, P = 0.027) as well as total muscle concentrations of lysine (r = 0.87, P = 0.05) for the weanling horses.
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Figure 5. Correlations between intake of leucine on a BW basis per day and plasma leucine concentrations (r = 0.93, P = 0.021) as well as total muscle concentrations (r = 0.88, P = 0.046) for the weanling horses.
plasma and free muscle amino acids along with the positive correlation between muscle and milk amino acid concentrations seems to support the priority for amino acid supply to milk. The WH group had several correlations for intake and plasma amino acids concentrations including threonine (r = 0.90, P = 0.036; Figure 3), lysine (r = 0.92, P = 0.027; Figure 4), valine (r = 0.89, P = 0.042), isoleucine (r = 0.96, P = 0.0083), leucine (P = 0.021; Figure 5), arginine (r = 0.93, P = 0.021), methionine (r = 0.89, P = 0.042; Figure 6), and phenylalanine (r = 0.90, P = 0.035; Figure 7). Intake and total muscle amino acids concentrations were correlated in the WH groups for phenylalanine (r = 0.88, P = 0.049; Figure 7), lysine (r = 0.87, P = 0.05; Figure 4), threonine (r = 0.90, P = 0.035; Figure 3), methionine (r = 0.87, P = 0.05; Figure 6), and leucine (r = 0.88, P
Figure 6. Correlations between intake of methionine on a BW basis per day and plasma concentrations of methionine (r = 0.89, P = 0.042) as well as total muscle concentrations of methionine (r = 0.87, P = 0.05) for the weanling horses.
Graham-Thiers et al.
Figure 7. Correlations between intake of phenylalanine on a BW basis per day and plasma concentrations of phenylalanine (r = 0.90, P = 0.035) as well as total muscle concentrations of phenylalanine (r = 0.88, P = 0.049) for the weanling horses.
Figure 9. Correlation of plasma concentrations of phenylalanine and total muscle concentrations of phenylalanine for the weanling horses (r = 0.92, P = 0.028).
= 0.046; Figure 5). Plasma and total muscle amino acids concentrations were correlated for lysine (r = 0.99, P = 0.0013; Figure 8), phenylalanine (r = 0.92, P = 0.028; Figure 9), and leucine in the WH group (r = 0.98, P = 0.0034; Figure 10). The large number of correlations between plasma and muscle amino acid concentrations observed in the WH group support the conclusion that the supply of amino acids closely reflected the subsequent demand for those amino acids by the growing horse. This could also be indicative of an anabolic state in which growth would be considered an anabolic function. The EH group had a negative correlation between total muscle amino acid concentrations and free amino acid muscle concentrations of histidine (r = −0.88, P = 0.0098) and between plasma and total muscle concentrations of phenylalanine (r = −0.82, P = 0.024; Figure 11). As stated previ-
ously, negative correlations between amino acid concentrations in plasma and muscle may suggest that other tissues may be taking priority over muscle tissue in the current function. This would not seem to be warranted in the exercising horse and may suggest catabolic state for the EH when the samples were taken. This is a limitation to taking single samples and admittedly may not represent the ongoing state of the EH. The MH group had negative correlations between intake and free muscle amino acid concentrations of lysine (r = −0.74, P = 0.037) as well as between plasma and total muscle amino acid concentrations of arginine (r = −0.89, P = 0.003; Figure 12). No other correlations were observed. The lack of correlations in the MH group could therefore also support the conclusion that the amino acid supply did not reflect the demand by the MH group, which is also supported by the large increases in plasma amino acid
Figure 8. Correlation of plasma concentrations of lysine and total muscle concentrations of lysine for the weanling horses (r = 0.99, P = 0.0013).
Figure 10. Correlation of plasma concentrations of leucine and total muscle concentrations of leucine for the weanling horses (r = 0.98, P = 0.0034).
Amino acids in horses in various functions
Fuller, M. F., R. McWilliam, T. C. Wang, and L. R. Giles. 1989. The optimum dietary amino acid pattern for growing pigs. 2. Requirements for maintenance and tissue accretion. Br. J. Nutr. 62:255–267.
Table 7. Total and free milk amino acids for LH mares at d 30 of lactation Amino acid Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine 1 2
Free amino acids, mmol/L
Total amino acids, mmol/L
Ratio1
0.07 ± 0.015 0.30 ± 0.065 0.09 ± 0.010 0.19 ± 0.040 0.12 ± 0.025 ND2 0.10 ± 0.025 0.11 ± 0.017 0.42 ± 0.050
25.1 ± 1.3 12.9 ± 0.7 26.8 ± 1.9 54.5 ± 2.8 40.2 ± 2.6 10.9 ± 0.6 20.1 ± 1.1 32.3 ± 1.8 37.6 ± 1.6
62 32 67 136 100 27 50 80 94
Lysine set at 100; all others compared with lysine. ND = not detected.
IMPLICATIONS
Figure 12. Correlation between plasma concentrations of arginine and total muscle concentrations of arginine for the maintenance horses (r = −0.89, P = 0.003).
concentrations in this group postfeeding, thus suggesting excesses. The free and total milk amino acids are reported in Table 7. The total milk amino acid ratios were calculated in relation to lysine. These ratios
Graham-Thiers, P. M., J. A. Wilson, J. Haught, and M. Goldberg. 2010a. Case Study: Relationships between dietary, plasma, and muscle amino acids in maintenance horses, exercising horses, and growing weanling horses. Prof. Anim. Sci. 26:313–319. Graham-Thiers, P. M., J. A. Wilson, J. Haught, and M. Goldberg. 2010b. Case Study: Relationships between dietary, plasma, and muscle amino acids in maintenance, pregnant, and lactating mares. Prof. Anim. Sci. 26:320–327. Henneke, D. R., G. D. Potter, J. L. Kreider, and B. F. Yeates. 1983. Relationship between condition score, physical measurements and body fat percentage in mares. Equine Vet. J. 15:371–372.
are in good agreement with other published reports of milk amino acid ratios (Bryden, 1991; Wickens et al., 2002).
Figure 11. Correlation of plasma concentrations of phenylalanine and total muscle concentrations of phenylalanine for the exercising horses (r = −0.82, P = 0.024).
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It appears from these data that for the most part, the body does not alter the muscle amino acid pattern regardless of the demands on the body. The supply of amino acids from plasma and muscle free amino acids does vary depending on function (as well as intake). The authors want to reiterate, however, that the data of the current study provide information only for the time point in which the plasma was sampled. The data may appear different if taken at another time point. These data provide additional information regarding amino acids in the equine, particularly the total muscle amino acid profile as well as the milk amino acid profile for which there is limited data. These data could assist in the development of an ideal protein concept for the horse.
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Miller-Graber, P. A., L. M. Lawrence, E. Kurcz, R. Kane, K. Bump, M. Fisher, and J. Smith. 1990. The free amino acid profile in the middle gluteal before and after fatiguing exercise in the horse. Equine Vet. J. 22:209–210. NRC. 2001a. Nutrient Requirements of Dairy Cattle. 7th ed. Natl. Acad. Press, Washington, DC. NRC. 2001b. Nutrient Requirements of Swine. 10th ed. Natl. Acad. Press, Washington, DC. NRC. 2007. Nutrient Requirements of Horses. 6th ed. Natl. Acad. Press, Washington, DC. Ott, E. A., R. L. Asquith, J. P. Feaster, and F. G. Martin. 1979. Influence of protein level and quality on growth and development of yearling foals. J. Anim. Sci. 49:620–626. Potter, E. L., D. B. Purser, and W. G. Bergen. 1972. A plasma reference index for predicting limiting amino acids of sheep and rats. J. Anim. Sci. 34:660–671. Russell, M. A., A. V. Rodiek, and L. M. Lawrence. 1986. Effect of meal schedules and fasting on selected plasma free amino acids in horses. J. Anim. Sci. 63:1428–1431. Sewell, D. A., R. C. Harris, and M. Dunnett. 1991. Carnosine accounts for most of the variation in the physic-chemical buffering in equine muscle. Equine Exercise Physiol. 3:276–280. Sewell, D. A., R. C. Harris, D. J. Marlin, and M. Dunnett. 1992. Estimation of the carnosine content of different fibre types in the middle gluteal muscle of the Thoroughbred horse. J. Physiol. 455:447–453. Wickens, C. L., P. K. Ku, and N. L. Trottier. 2002. An ideal protein for the lactating mare. J. Anim. Sci. 80(Suppl. 1):155.